Our goal during the past thirty years of NIH funding has been to delineate the mechanism of chemotaxis in the Gram-positive bacterium Bacillus subtilis. During the past decade, the realization has emerged that the B. subtilis mechanism might be the ideal paradigm for understanding the mechanism in the broad sweep of Bacteria and Archaea since it is so similar to that in the Archaea. It is an ancient mechanism and close to the progenitor mechanism that existed just before the separation of the Bacteria and Archaea. Thus in studying this mechanism, we can see where the current mechanisms in Bacteria and Archaea came from and get insights into how they might currently work. The mechanism involves two proteins, found in the Archaea but not in Escherichia coli CheC and CheD. It also involves receptors that, like those in the Archaea, have two pairs of insertions of four turns of n-helix in two locations in the cytoplasmic region of the receptors, compared with what is found in E. coll. It also involves another protein CheV, found only in Bacteria but very widespread there, although not in E. coil. The B. subtilis mechanism has a large switch protein, FliY, whose C-terminal region is homologous to the small E. coli protein FliN but no one knew why the protein was so large. During the last funding period biochemical events associated with each of the novel proteins or with the receptor differences between B. subtilis and E. coli have been described so that the usefulness of the B. subtilis mechanism as a paradigm has become much enhanced. CheC and FliY are CheY-P phosphatases, CheD is a deamidase and stimulates CheC, whereas CheC inhibits CheD. CheV brings about adaptation by becoming phosphorylated. Methylation of receptors at nearby sites has opposite effects on the kinase. At this point, we wish to take our quest to understand this mechanism to a new level, namely, to refine our work on individual proteins and to understand how the various chemotaxis proteins interact to bring about excitation and adaptation--that is, chemotaxis. We have developed the genetic stocks and in vitro assays to do this. We propose to map out where each of the proteins interacts with the others and, using mutants, show the function of each interaction. We propose detailed characterization of receptor methylation to pin down the roles of each site. We propose experiments to clarify the roles of each of the enzymatic and binding functions of CheC, CheD, and CheV.